Due to significant improvements in the early detection and the treatment of cancer, the number of cancer survivors is increasing. Unfortunately, long-term cancer survivors are at increased risk for late effects related to cancer treatment, including ionizing radiation (IR)- and chemotherapy-induced long-term bone marrow (LT-BM) injury. LT-BM injury is primarily attributed to selective induction of oxidative stress and senescence in hematopoietic stem cells (HSCs). The goals of this project are to use our well characterized LT-BM injury mouse models induced by total body irradiation (TBI) or chemotherapy to (1) identify the primary cellular origins of reactive oxygen species (ROS) in HSCs, (2) elucidate how production of ROS in HSCs is regulated, and (3) develop a mechanism-based strategy that can effectively inhibit IR- and chemotherapy-induced HSC senescence and LT-BM suppression via selective inhibition of ROS production in HSCs. Specifically, the hypotheses to be tested are that (1) ROS derived from NADPH oxidase 4 (NOX4) is primarily responsible for mediating IR- and chemotherapy-induced HSC senescence and (2) inhibition of NOX4 can be developed as a novel therapeutic strategy to effectively reduce IR- and chemotherapy-induced LT-BM injury. Three specific aims will be pursued to test these hypotheses. Aim 1: Determine whether NOX4 is primarily responsible for the production of ROS in HSCs to mediate TBI-induced HSC senescence and LT-BM injury. Aim 2: Investigate whether activation of the mammalian target of rapamycin (mTOR)-hypoxia-inducible factor 1 (HIF1) pathway mediates IR-induced upregulation of NOX4 in HSCs. Aim 3: Determine whether inhibition of NOX4 activity with a NOX inhibitor or suppression of NOX4 upregulation with an mTOR inhibitor can effectively inhibit HSC senescence and LT-BM injury induced by not only IR but also chemotherapy. These proposed studies will gain fundamental insights into the pathogenesis of LT-BM injury induced by IR and chemotherapy. These insights will aid in the development of more effective strategies to prevent and mitigate IR- and chemotherapy-induced LT-BM toxicity. Because increased expression of NOX4 and aberrant activation of mTOR are involved in tumorigenesis and tumor resistance to therapy, targeted inhibition of NOX4 or mTOR may provide cancer patients the dual benefits of reducing IR- and chemotherapy-induced LT-BM suppression while increasing tumor response to cancer therapy.